Open AccessThe amino terminus regulates binding to and activation of cGMP‐dependent protein kinase
Author(s) -
LANDGRAF Wolfgang,
HOFMANN Franz
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb14771.x
Subject(s) - allosteric regulation , protein kinase a , biochemistry , enzyme , kinase , chemistry , protein subunit , cgmp dependent protein kinase , binding site , phosphorylation , enzyme activator , pde10a , stereochemistry , biophysics , biology , mitogen activated protein kinase kinase , phosphodiesterase , gene
The allosteric regulation of binding to and the activation of cGMP‐dependent protein kinase (cGMP kinase) was studied under identical conditions at 30°C using three forms of cGMP‐kinase which differed in the aminoterminal segment, e.g. native cGMP kinase, phosphorylated cGMP kinase which contained 1.4±0.4 mol phosphate/subunit and constitutively active cGMP kinase which lacked the amino‐terminal dimerization domain. These three enzyme forms have identical kinetic constants, e.g. number of cGMP‐binding sites, K m values for MgATP and the heptapeptide Kemptide, and V max values. In the native enzyme, MgATP decreases the affinity for binding site 1. This effect is abolished by 1 M NaCl. In contrast, high concentrtions of Kemptide increase the affinity of binding site 2 about fivefold. Under the latter conditions, identical K d values of 0.2 μM were obtained for sites 1 and 2. Salt, MgATP and Kemptide do not affect the binding kinetics of the phosphorylated or the constitutively active enzyme, suggesting that allosteric regulation depends solely on the presence of a native amino‐terminal segment. Cyclic GMP activates the native enzyme at K a values which are identical with the K d values for both binding sites. The activation of cGMP‐dependent protein kinase is noncooperative but the K a value depends on the substrate peptide concentration. These results show that the activity of cGMP kinase is primarily regulated by conformational changes within the amino‐terminal domain.